JP2009100780A - Mammalian gene involved in viral infection and tumor suppression - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide methods of identifying cellular genes necessary for viral growth and cellular genes that function as tumor suppressors. <P>SOLUTION: The problem is solved by providing a gene having a sequence disclosed in the sequence table of the invention. Provided are nucleic acids for reducing or preventing viral infection or cancer, and methods thereof. Also provided are methods of producing substantially virus-free cell cultures and methods for screening such genes. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  This invention was made with government support in part under grants from the National Institutes of Health grant number CA68283 and the Department of Veterans Affairs. The United States government has certain rights in this invention.

(Field of Invention)
The present invention provides a method for identifying cellular genes used for viral growth or tumor progression. Accordingly, the present invention relates to nucleic acids and methods therefor associated with reducing or preventing viral infection and inhibiting tumor progression. The invention also relates to a method for screening for additional such genes.

(Background technology)
Various projects have been devoted to isolating and sequencing the genomes of various animals, especially humans. However, most methodologies provide nucleotide sequences whose functions are not related or even suggested, thus limiting the immediate usefulness of such data.

  The present invention, in contrast, provides a method of screening only for nucleic acids involved in a particular process (ie, viral infection or tumor progression). For viral infections, isolated nucleic acids are useful in the treatment of these processes. This is because only nucleic acids that are not necessary for the cell are isolated by this method. Such a method is very useful. This is because the method is attributed to the function of each isolated gene, and thus the isolated nucleic acid can be readily utilized in a variety of specific methods and procedures.

  For example, the present invention provides a method for isolating a nucleic acid encoding a gene product that is used for viral infection but is not required for a cell. Viral infection is a significant cause of human morbidity and mortality. Understanding the molecular mechanism of such infection leads to new approaches in these treatments and controls.

  Viruses can establish various types of infections. Although these infections can generally be classified as lytic or persistent, some lytic infections are considered persistent. In general, persistent infections fall into two categories: (1) Chronic (productive) infections (ie, infectious viruses are present and recovered by traditional biological methods And (2) latent infections (ie, infections in which the viral genome is present in the cell, but infectious viruses are generally not produced except during intermittent episodes of reactivation). Persistence generally includes both productive and latent infection states.

  A lytic infection can also persist under conditions in which only a small portion of all cells are infected (smolder (circulating) infection). Although few infected cells release the virus and are killed, the progeny virus again infects only a small number of total cells. Examples of such decubitus infection include lactate dehydrogenase virus in mice (Non-patent Document 1) and adenovirus infection in humans (Porter, DD 784-790, edited by Baron, S, Medical Microbiology No. 2). Persistence of the edition (Addison-Wesley, Menlo Park, CA 1985).

  Furthermore, the virus may be lytic for some cell types but not lytic for other cell types. For example, evidence shows that human immunodeficiency virus (HIV) is more lytic to T cells than monocytes / macrophages and thus can result in productive infection of T cells that can result in cell death. However, it suggests that HIV-infected mononuclear phagocytes can produce virus without lysis for a considerable period of time. (Klatzmann et al., Science 225: 59-62 (1984); Koyanagi et al., Science 241: 1673-1675 (1988); Sattentau et al., Cell 52: 631-633 (1988)).

  Traditional treatments for viral infections include pharmaceuticals aimed at specific virus-derived proteins such as HIV protease or reverse transcriptase, or recombinant (cloned) immune modulators (host-derived) such as interferons. However, current methods have some limitations and drawbacks, including a high percentage of viral mutations that make antiviral formulations ineffective. For immune modulators, limited efficacy, limiting side effects, lack of specificity all limit the general applicability of these agents. Also, the rate of success with current antiviral and immune modulators is disappointing.

  One aspect of the present invention focuses on isolating genes that are not essential for cell survival when disrupted in one or both alleles, but are required for viral replication. This can occur with dose effects, where one allelic knockout can confer a virus resistant phenotype to the cell. As targets for therapeutic intervention, these cellular gene products (proteins, portions of proteins (modified enzymes including but not limited to glycation, lipid modifications such as myriolate)), lipids, transcription elements, and Inhibition (including RNA regulatory molecules) appears to have few strong toxic side effects, and viral mutations appear to overcome little “blocking” to successful replication.

  The present invention provides a significant improvement over previous methods of therapeutic intervention attempted against viral infection by addressing the cellular genes required for the virus to grow. Thus, the present invention also provides a breakthrough therapeutic approach for intervening in viral infection by providing a method of treating virus by inhibiting cellular genes required for viral infection. Since these genes are not required for cell survival at the level of expression required to inhibit viral replication by the means by which they were originally detected, these treatment methods appear to have been found in previous methods. It can be used on a subject without causing serious adverse effects on the subject. The present invention also provides the surprising discovery that virally infected cells depend on factors in the serum to survive. Thus, the present invention also provides a method for treating viral infections by inhibiting this serum survival factor. Finally, these findings also remove virally infected cells from cell culture by removing, inhibiting or destroying this serum survival factor in the culture so that uninfected cells selectively survive. Provide a new way to remove.

  The selection of tumor suppressor gene (s) has become an important area in the discovery of new targets for therapeutic intervention in cancer. Since the discovery that certain genes that can suppress the “transformed” phenotype limit cells from indiscriminate entry into the cell cycle, considerable time has been spent in the discovery of such genes. Yes. Some of these genes include the gene associated with rhabdomyosarcoma (Rb) and the gene associated with the p53 (apoptosis-related) coding gene. The present invention uses gene traps to select cell lines having a phenotype transformed from untransformed cells and to suppress malignant or transformed phenotypes from these cells. A method for isolating the resulting gene is provided. Thus, the function of the isolated gene is related to the characteristics of the isolation process. The ability to rapidly select tumor suppressor genes may provide a unique target in the process of treating or preventing cancer and further diagnostic testing.

Mahy, B .; W. J. et al. , Br. Med. Bull. 41: 50-55 (1985)

The present invention provides, for example:
(Item 1) An isolated nucleic acid comprising a nucleotide sequence as described below:
Sequence number 1, Sequence number 2, Sequence number 3, Sequence number 5, Sequence number 6, Sequence number 7, Sequence number 8, Sequence number 9, Sequence number 10, Sequence number 11, Sequence number 12, Sequence number 13, Sequence number 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 6 SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 , SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, Sequence SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126 Preliminary SEQ ID NO: 127.
(Item 2) A nucleic acid comprising at least 20 consecutive nucleotides of the nucleotide sequence according to item 1.
(Item 3) A nucleic acid comprising at least 30 contiguous nucleotides of the nucleotide sequence according to item 1.
(Item 4) A nucleic acid comprising at least 40 contiguous nucleotides of the nucleotide sequence according to item 1.
(Item 5) An isolated nucleic acid encoding a protein encoded by a gene comprising the nucleotide sequence described below:
Sequence number 1, Sequence number 2, Sequence number 3, Sequence number 5, Sequence number 6, Sequence number 7, Sequence number 8, Sequence number 9, Sequence number 10, Sequence number 11, Sequence number 12, Sequence number 13, Sequence number 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 6 SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 , SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, Sequence SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126 Preliminary SEQ ID NO 127 or their homologs.
(Item 6) A host cell comprising the nucleic acid according to item 1 or 5.
(Item 7) A nucleic acid comprising a nucleic acid that selectively hybridizes under stringent conditions with the nucleic acid according to item 1 or 5.
(Item 8) A nucleic acid having a region within an exon, wherein the region has at least 50% homology with the nucleic acid according to item 1 or 5.
(Item 9) A nucleic acid having a region within an exon, wherein the region has at least 60% homology with the nucleic acid according to item 1 or 5.
(Item 10) A nucleic acid having a region within an exon, wherein the region has at least 70% homology with the nucleic acid according to item 1 or 5.
(Item 11) A nucleic acid having a region within an exon, wherein the region has at least 80% homology with the nucleic acid according to item 1 or 5.
(Item 12) A nucleic acid having a region within an exon, wherein the region has at least 90% homology with the nucleic acid according to item 1 or 5.
(Item 13) A nucleic acid having a region within an exon, wherein the region has at least 95% homology with the nucleic acid according to item 1 or 5.
(Item 14) A polypeptide comprising an amino acid sequence encoded by the nucleic acid according to item 1 or 5.
(Item 15) A nucleic acid comprising a regulatory region of a gene comprising the nucleotide sequence described below:
Sequence number 1, Sequence number 2, Sequence number 3, Sequence number 5, Sequence number 6, Sequence number 7, Sequence number 8, Sequence number 9, Sequence number 10, Sequence number 11, Sequence number 12, Sequence number 13, Sequence number 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 6 SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 , SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, Sequence SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126 Preliminary SEQ ID NO 127 or their homologs.
(Item 16) A construct comprising the regulatory region of item 15, wherein the regulatory region is operably linked to a reporter gene.
(Item 17) A method for reducing or inhibiting viral infection in a subject comprising the nucleic acids described below:
Sequence number 1, Sequence number 2, Sequence number 3, Sequence number 4, Sequence number 5, Sequence number 6, Sequence number 7, Sequence number 8, Sequence number 11, Sequence number 12, Sequence number 13, Sequence number 14, Sequence number 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, Sequence number 32, sequence number 33, sequence number 34, sequence number 35, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 41, sequence number 42, sequence number 43, sequence number 44, sequence number 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 5 SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, Sequence No. 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 1 4, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126 or SEQ ID NO: 127, or homologues thereof A method comprising administering an amount of a composition that inhibits expression or functionalization of a gene product encoded by the gene, thereby treating the viral infection.
(Item 18) The method according to item 17, wherein the gene is selected from a nucleic acid encoding a gene product derived from the group consisting of: or the gene product is selected from the group consisting of: ,Method:
Tristetraproline (human ZFP-36), 6-pyruvoyltetrahydropterin synthase, eukaryotic DnaJ-like protein, ID3 (inhibitor of DNA binding 3), N-acetylglucosaminyltransferase I (mGAT-1), cleavage Stimulating factor (CSTF2), TAK1 binding protein, human zinc transcription factor ZPF207, Dlx2, Smad7 (Mad related protein), and P glycoprotein (mdr1b).
(Item 19) The method according to item 17, wherein the subject is a human.
(Item 20) A method for reducing or inhibiting viral infection in a subject, wherein the selected nucleic acid is a nucleic acid described below:
Sequence number 1, Sequence number 2, Sequence number 3, Sequence number 4, Sequence number 5, Sequence number 6, Sequence number 7, Sequence number 8, Sequence number 11, Sequence number 12, Sequence number 13, Sequence number 14, Sequence number 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, Sequence number 32, sequence number 33, sequence number 34, sequence number 35, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 41, sequence number 42, sequence number 43, sequence number 44, sequence number 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 5 SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, Sequence No. 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 1 4, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126 or SEQ ID NO: 127, or homologues thereof Exogenously mutating an endogenous gene into a mutant gene that cannot produce a functional gene product of the gene or a mutant gene that produces a reduced amount of the functional gene product of the gene, and in the subject Placing the cell, thereby reducing viral infection of the cell in the subject. Let's do it.
(Item 21) The method according to item 20, wherein the cell is a hematopoietic cell.
(Item 22) The method according to item 20, wherein the subject is a human.
(Item 23) The method according to item 20, wherein the cell is derived from the subject.
24. A method for screening compounds for efficacy in treating or preventing viral infection, comprising:
Sequence number 1, Sequence number 2, Sequence number 3, Sequence number 4, Sequence number 5, Sequence number 6, Sequence number 7, Sequence number 8, Sequence number 11, Sequence number 12, Sequence number 13, Sequence number 14, Sequence number 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, Sequence number 32, sequence number 33, sequence number 34, sequence number 35, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 41, sequence number 42, sequence number 43, sequence number 44, sequence number 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 5 SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, Sequence No. 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 1 4, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, The nucleic acid described in SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126 or SEQ ID NO: 127, or a homologue thereof And administering the compound to a cell containing a cellular gene that functionally encodes a cellular gene comprising: and a gene product that is necessary for replication of the virus in the cell but not for the survival of the cell And detecting the level and / or activity of the gene product produced A method wherein a reduction or elimination of the gene product and / or gene product activity is indicative of a compound that is effective for treating or preventing the viral infection.
25. A method of screening a compound for reducing or inhibiting viral infection, comprising administering the compound to a cell comprising the construct of item 16, and of the produced reporter gene product. Detecting a level, wherein the reduction or elimination of the reporter gene product indicates a compound for reducing or inhibiting the viral infection.
26. A method for screening compounds for efficacy in treating or preventing viral infection, comprising:
Sequence number 1, Sequence number 2, Sequence number 3, Sequence number 4, Sequence number 5, Sequence number 6, Sequence number 7, Sequence number 8, Sequence number 11, Sequence number 12, Sequence number 13, Sequence number 14, Sequence number 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, Sequence number 32, sequence number 33, sequence number 34, sequence number 35, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 41, sequence number 42, sequence number 43, sequence number 44, sequence number 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 5 SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, Sequence No. 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 1 4, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, or the nucleic acid described in SEQ ID NO: 127, or Contacting a gene product of a cellular gene containing a homolog with the compound, and detecting the function of the gene product, wherein reducing or eliminating the function is for reducing or inhibiting viral function A method showing a compound.
(Item 27) A method for suppressing a malignant phenotype in a cell in a subject, comprising: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: Administering a quantity of a composition that inhibits the expression or functionalization of the gene product encoded by the gene comprising the nucleic acid set forth in SEQ ID NO: 36 or SEQ ID NO: 94, or a homologue thereof, whereby malignant expression Method to suppress the mold.
28. A method of screening a compound for efficacy in treating a viral infection, comprising administering the compound to a cell containing a cellular gene that functionally encodes a gene product, wherein And wherein the overexpression of the gene product inhibits the replication of the virus but does not prevent survival of the cell, and detecting the level of the gene product produced, wherein increasing the gene product comprises A method showing compounds effective in the treatment of viral infections.
(Item 29) A method for screening for a compound capable of suppressing a malignant phenotype in a cell, comprising: Administering to a cell containing a nucleic acid functionally encoding a gene product encoded by a gene comprising the nucleic acid set forth in SEQ ID NO: 36 or SEQ ID NO: 94, or a homologue thereof, and the level of the gene product produced Wherein the increase in the gene product indicates a compound effective to suppress the malignant phenotype.

(Detailed description of the invention)
The present invention utilizes a “gene trap” method with a selection process for identifying and isolating nucleic acids from genes associated with specific functions. Specifically, it provides a means for isolating cellular genes that are necessary for viral infection but are not essential for cell survival, and a means for isolating cellular genes that suppress tumor progression.

  The present invention also provides a core discovery that virally infected cells become dependent on at least one factor present in the serum for survival, while uninfected cells do not exhibit this dependency. This core discovery has been utilized in many ways in the present invention. Initially, inhibition of “serum survival factor” can be utilized to annihilate persistent virally infected cells from a population of uninfected cells. Inhibition of this factor can also be used to treat a viral infection in a subject, as further described herein. Furthermore, inhibition or recovery of serum survival factors in tissue culture allows detection of cellular genes that are required for viral replication but are not essential for the survival of uninfected cells. The present invention further provides several such cellular genes, as well as methods for treating viral infections by inhibiting the function of such genes.

  Furthermore, the present invention provides cellular genes whose overexpression is associated with inhibition of viral growth and / or replication.

  The method provides a number of cellular genes that are necessary for viral growth in the cell but are not essential for cell survival. These genes are important for viral lytic and persistent infection. These genes create a gene trap library by infecting cells with a retroviral gene trap vector and a gene trap event occurs (ie, the vector is inserted, resulting in the insertion of a promoter-free marker gene, resulting in cells Select cells where the sex promoter promotes transcription of the marker gene (ie, is inserted into a functioning gene), serum starve the cells, and infect selected cells with the selected virus during continuous serum starvation, It was then isolated by adding serum to allow color development of visible colonies cloned by limiting dilution. The gene inserted into the retroviral gene trap vector was then isolated from the colony using a probe specific for the retroviral gene trap vector. Thus, the nucleic acid isolated by this method is part of the isolated gene. In addition, using this method, several cellular genes were isolated where overexpression prevented viral infection or tumor growth. And they provide a way to treat viral infection or tumor growth / suppression by overexpression of these genes.

  Accordingly, the present invention provides a method of identifying cellular genes that are necessary for viral growth in a cell and are not essential for cell survival, the method comprising: (a) a cell culture (eg, a serum-containing medium) A step of transferring a vector encoding a selective marker gene lacking a functional promoter, (b) selecting a cell expressing the marker gene, and (c) removing serum from the culture medium (D) isolating a cell culture with the virus; and (e) isolating a cellular gene into which the marker gene has been inserted from living cells, thereby being necessary for virus propagation in the cell; and Identifying a gene that is not essential for cell survival. The present invention also provides a method for identifying cellular genes that are used for virus growth in cells and are not essential for cell survival, which method (a) functions in cell cultures grown in serum-containing media. A step of gene transfer with a vector encoding a selective marker gene lacking a specific promoter, (b) a step of selecting a cell expressing the marker gene, (c) a step of removing serum from the culture medium, (d) a cell Infecting the culture with virus, and (e) isolating a cellular gene from which the marker gene has been inserted from living cells, thereby being necessary for virus growth in the cell and not essential for cell survival Identifying a gene, or a gene whose overexpression prevents viral replication but is not critical to cell survival. It can be readily determined whether serum starvation is required for selection in any selected cell type (eg, Chinese hamster ovary cells). Otherwise, serum starvation can be excluded from this process.

  Alternatively, instead of removing serum from the culture medium, serum factors required for virus growth can be inhibited, for example, by administration of antibodies that specifically bind to the factors. Furthermore, if no persistently infected cells are present in the culture, the serum starvation step can be omitted and the cells can be grown in normal media for the cell type. If serum starvation is used, it can be continued for a period of time after the culture is infected with the virus. Serum can then be added to the culture. If some other method is used to inactivate the factor, it can be stopped, inactivated, or removed (eg, this anti-factor) before adding fresh serum to the culture. The antibody can be removed (eg, using a binding antibody directed against the antibody). Surviving cells are mutants with inactivated inserts in genes required for viral growth. The gene with the insert can then be isolated by isolating the sequence with the marker gene sequence. This process of mutation prevents wild type function. Mutant genes can produce normal products at low levels, can produce normal products that are not normally found in these cells, can cause normal products to overproduce, May produce a modified product that has the functions of but not others, or may completely disrupt the gene function. Furthermore, mutations can destroy RNA that has a function, but is never translated into protein. For example, the alpha tropomyosin gene has a 3 'RNA that is very important for cellular regulation but never translated into protein. (Cell 75 1107-1117, December 17, 1993).

As used herein, a cellular gene “not essential for cell survival” means a gene in which disruption of one or both alleles results in a cell that is viable for at least a period of time; This makes it possible to inhibit viral replication for prophylactic or therapeutic use or for research use. A gene “necessary for viral growth” is the gene product of either protein or RNA, secreted or not, either directly or indirectly in some ways for the virus to grow. In the absence of its gene product (ie, a functionally available gene product), the virus does not spread. For example, such genes include cell cycle regulatory proteins, proteins that act in vacuolar hydrogen pumps, or protein folding and protein modification, including but not limited to: phosphorylation, methylation, glycation, myristylation Or may encode a protein involved in other lipid moieties, or protein processing via enzymatic processing). Some examples of such genes are vacuolar H ⁺ ATPase, alpha tropomyosin, gas5 gene, ras complex, N-acetyl-glucosaminyl-1-transferase I mRNA, annexin II, c-Golgi CM130, and cal Contains cyclin.

  Any virus that can infect cells can be used in this method. The virus can be selected based on the particular infection that is desired to be studied. However, it is contemplated by the present invention that many viruses are dependent on the same cellular gene for survival; therefore, cellular genes isolated using one virus are also therapeutic for other viruses. It can also be used as a target for. Any cellular gene can be determined using the methods described herein (ie, generally inhibiting a gene or its gene product in a cell and whether the desired virus can grow in that cell) Can be tested for relevance to any desired virus. Some examples of viruses include: HIV (including HIV-1 and HIV-2); parvovirus; papilloma virus; hantavirus; influenza virus (eg, influenza A, B, and C viruses); Hepatitis virus; calicivirus; astrovirus; rotavirus; coronavirus (eg, human respiratory coronavirus); picornavirus (eg, human rhinovirus and enterovirus); ebola virus; human herpesvirus (eg, HSV-1- 9); human adenovirus; for animals, animal counterparts of any of the above human viruses, animal retroviruses (eg simian immunodeficiency virus, avian immunodeficiency virus, bovine immunodeficiency virus, feline immunodeficiency virus) , Equine infection Anemia virus, caprine arthritis encephalitis virus, arenaviruses, arboviruses, tick borne virus or visna virus) may be mentioned.

  Nucleic acids containing the cellular genes of the present invention were isolated as described in the methods and examples above. The present invention includes nucleic acids comprising the nucleotide sequences set forth below: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 7, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, Sequence number 76, sequence number 77, sequence number 78, sequence number 79, sequence number 80, sequence number 81, sequence number 83, sequence number 84, sequence number 85, sequence number 86, sequence number 87, sequence number 88, sequence number 89, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, Sequence number 105, Sequence number 106, Sequence number 107, Sequence number 108, Sequence number 112, Sequence number 120, Sequence number 121, Sequence number 122, Sequence Issue 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126 and SEQ ID NO: 127 (This list is sometimes referred to as briefly "Sequence Listing 1" herein). Thus, these nucleic acids may contain additional nucleotides at either end of the molecule in addition to the nucleotides set forth in each SEQ ID NO in the sequence listing. Such additional nucleotides can be added by any standard method, such as recombinant and synthetic methods, as is known in the art. Examples of such nucleic acids comprising a nucleotide sequence as set forth in any item of the sequence listing contemplated by the present invention include, for example, nucleic acids placed in a vector; a nucleic acid having one or more regulatory regions (eg, promoter, enhancer, polyadenylation site) linked so that mRNA or protein can be produced; a nucleic acid comprising additional nucleic acids of this gene (eg, larger of this gene) Or full length genomic fragment, partial or full length cDNA, partial or full length RNA). Making and / or isolating such larger nucleic acids is further described below and is well known and standard in the art.

  Double-stranded nucleic acids corresponding to the nucleic acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 136 (including SEQ ID NO: 1 and SEQ ID NO: 136) are also provided in the present invention. As used herein, “nucleic acid” may refer to both or any strand of such a double stranded nucleic acid (the strands often referred to as “positive” and “negative” strands). Either strand of such a double stranded nucleic acid can encode a polypeptide of the invention, and the coding sequence of such a polypeptide can be translated along the direction of either strand. Examples of polypeptides encoded by either chain are disclosed herein.

  The invention also provides nucleic acids encoding proteins encoded by genes comprising nucleotide sequences set forth in any of the sequences listed in Sequence Listing 1, and allelic variants and homologues of each such gene . Genes are readily obtained using standard methods as described below and are known in the art and standard. The present invention also contemplates any unique fragments of these genes or any unique fragments of nucleic acids set forth in any sequence listed in Sequence Listing 1. Examples of fragments of the gene of the present invention may include nucleic acids having the sequence shown in any sequence listed in Sequence Listing 1. To be unique, this fragment is to be distinguished from other known sequences, most easily determined by comparing any nucleic acid fragment to the nucleotide sequence of the nucleic acid in a computer database such as GenBank. Must be of sufficient size. Such a comparative search is standard in the field. Typically, unique fragments that are useful as primers or probes are at least about 20 to about 25 nucleotides in length, depending on the specific nucleotide content of the sequence. Further, the fragment can be, for example, at least about 30, 40, 50, 75, 100, 200 or 500 nucleotides in length. The nucleic acid can be single-stranded or double-stranded, depending on the intended purpose.

  The present invention further provides a nucleic acid comprising a regulatory region of a gene comprising any one of the nucleotide sequences shown in Sequence Listing 1 and homologs of each such gene. Further provided are constructs comprising such regulatory regions operably linked to a reporter gene. Such a reporter gene construct can be used for screening for compounds and compositions that affect the expression of a gene comprising a nucleic acid having the sequence shown in Sequence Listing 1 or a homologue thereof.

The nucleic acids shown in the sequence listing are gene fragments; both the entire coding sequence and the entire gene including each fragment are contemplated herein, and by standard methods given the nucleotide sequences shown in the sequence listing Easily available (eg, Sambrook et al., Molecular Cloning:
A Laboratory Manual, 2nd edition, Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York, 1989; DNA Cloning: A Practical Approach, Volumes 1 and 2, Glover,
D. M.M. Ed., IRL Press Limited, Oxford, 1985). In order to obtain a whole genome gene, briefly, the nucleic acid whose sequence is shown in any of SEQ ID NOs: 1 to 127, or preferably any of the sequences shown in Sequence Listing 1, or a small fragment thereof, Under stringency conditions, a genomic library is utilized as a probe to screen and isolated clones are sequenced. Once the sequence of the new clone is determined, the probe can be devised from a portion that is not present in the previous fragment of the new clone, and more clones that hybridize with the library and contain the fragment of the gene. Can be isolated. Thus, by repeating this process in a systematic manner, one can “walk” along the chromosome and finally obtain the nucleotide sequence of the entire gene. Similarly, a portion of the fragment of the invention containing an open reading frame or an additional fragment obtained from a genomic library can be used to screen a cDNA library to obtain a cDNA having the entire coding sequence of the gene. . Repeated screening can be utilized as described above to obtain complete sequences from several clones as needed. The isolate can then be sequenced by standard means such as the dideoxynucleotide sequencing method to determine the nucleotide sequence (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring (See Harbor Laboratory, Cold Spring Harbor, New York, 1989).

  This gene was isolated from rats. However, homologues in any desired species (preferably a mammal, such as a human) can be obtained from a human library (genome or cDNA) using a probe comprising the sequence of the nucleic acid or fragment thereof shown in the sequence listing herein. And is preferably obtained by isolating a gene that specifically hybridizes with the probe, preferably under relatively high stringency hybridization conditions. For example, high salt conditions (eg, in 6 × SSC or 6 × SSPE) and / or high temperature hybridization can be used. For example, the stringency of hybridization is typically about 5 ° C. to about 20 ° C. below the Tm (melting point at which half of the molecules dissociate from the pair) for a given chain length. As is known in the art, the nucleotide composition of the hybridizing region is a factor in determining the melting point of the hybrid. For example, the recommended hybridization temperature for a 20mer probe is typically about 55-58 ° C. In addition, the rat sequence can be arbitrarily identified by determining the amino acid sequence for a portion of the rat protein and selecting a probe that optimizes the codon usage encoding the amino acid sequence of the homolog in a particular animal. Can be used to devise probes for homologues in other animals. Any isolated gene can sequence the gene and determine that the gene includes the nucleotide sequence listed herein as comprising the gene. Any homolog can be identified as the target gene by confirming its functionality as a homolog.

  Further, according to the present invention, 98%, 95%, 90% to the region in the exon of a nucleic acid encoding a protein encoded by a gene comprising a nucleotide sequence represented by any of the sequences listed in Sequence Listing I or a homologue thereof. Further contemplated are nucleic acids derived from any desired species, preferably mammals, and more preferably humans having%, 85%, 80%, 70%, 60%, or 50% or greater homology in the region of homology. The The present invention also relates to 98%, 95%, 90% in the homologous region relative to the region in the exon of the nucleic acid containing a nucleotide sequence represented by any of the sequences listed in Sequence Listing 1 of the Sequence Listing or a homologue thereof. Also contemplated are nucleic acids from any desired species, preferably mammals, more preferably humans, having 85%, 80%, 70%, 60% or 50% or greater homology. These genes can be synthesized or can be obtained by the same methods used to isolate homologs. This method can be used as a string of hybridization and washing, if desired, as the desired homology is reduced, and further depending on the GC or AT richness of any region where variability is sought. The gency is reduced. Any allelic variant of this gene or a homologue thereof can be easily isolated and sequenced by screening additional libraries according to the protocol described above. Methods for making synthetic genes are described in US Pat. No. 5,503,995 and references cited therein.

  The nucleic acid encoding any selected protein of the invention can be any nucleic acid that functionally encodes the protein. For example, to functionally encode (ie, allow expression of a nucleic acid), the nucleic acid can be expressed, for example, by exogenous or endogenous expression control sequences (eg, origins of replication, promoters, enhancers, and necessary information). Processing sites (eg, ribosome junctions, RNA splice sites, polyadenylation sites and transcription termination sequences)). A preferred expression control sequence may be a promoter derived from a metallothionein gene, an actin gene, an immunoglobulin gene, CMV, SV40, adenovirus, bovine papilloma virus, and the like. Expression control sequences can be selected for functionality in the cell in which the nucleic acid is placed. The nucleic acid encoding the selected protein can be readily determined based on the amino acid sequence of the selected protein, and clearly many nucleic acids encode any selected protein.

  The present invention is selective under stringent conditions with a nucleic acid encoding a nucleic acid described in Sequence Listing 1 or a protein encoded by a gene containing a nucleotide sequence shown in any sequence listed in Sequence Listing 1. Nucleic acids that hybridize to are further provided. This hybridization can be specific. The degree of complementarity between the hybridizing nucleic acid and the sequence to which the nucleic acid hybridizes should be at least sufficient to eliminate hybridization with a nucleic acid encoding an irrelevant protein. Thus, a nucleic acid that selectively hybridizes with a nucleic acid of the protein coding sequence does not selectively hybridize with a nucleic acid for a different, unrelated protein under stringent conditions, and vice versa. Typically, the stringency of hybridization to achieve selective hybridization is a high ionic strength solution at a temperature about 12-25 ° C. below Tm (the melting temperature at which half molecules dissociate from the pair). Including a wash at a combination of temperature and salt concentration selected such that the wash temperature following hybridization with (6 × SSC or 6 × SSPE) is about 5-20 ° C. lower than the Tm of the hybrid molecule. Temperature and salt conditions are readily determined empirically in preliminary experiments where a sample of reference DNA immobilized on a filter is hybridized to the labeled nucleic acid of interest and then washed under conditions of different stringency. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridization. The wash temperature can be used as described above to achieve selective stringency, as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al., 1989. 7: 7). Nucleic acid fragments that selectively hybridize to any given nucleic acid are used, for example, as primers and / or probes for further hybridization or amplification methods (eg, polymerase chain reaction (PCR), ligase chain reaction (LCR)). obtain. Preferred stringent hybridization conditions for DNA: DNA hybridization can be about 68 ° C. (in aqueous solution) in 6 × SSC or 6 × SSPE followed by a wash at 68 ° C.

The present invention further provides a polypeptide comprising an amino acid sequence encoded by a gene comprising the nucleotide sequence set forth in SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 21 SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 67 , SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, Sequence SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 , SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 08, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126 and SEQ ID NO: 127 (i.e., SEQ ID NO: 1). Further provided is a polypeptide comprising an amino acid sequence encoded by a nucleic acid that selectively hybridizes to the nucleic acid in Sequence Listing 1 under stringent conditions. In addition, the polypeptide comprises an amino acid sequence encoded by a nucleic acid having a region within the exon, which region has at least 50, 60, 70, 80, 90 or 95% homology with the nucleic acid in Sequence Listing 1. . These polypeptides can be readily obtained by any number of means. For example, the nucleotide sequence of the coding region of a gene can be translated and then the corresponding polypeptide can be mechanically synthesized by standard methods. In addition, the coding region of the gene can be expressed or synthesized, and specific antibodies to the resulting polypeptide can be raised by standard methods (eg, Harlow and Lane, Antibodies: A Laboratory Manual). , Cold Spring Harbor Laboratory, Cold Spring
(See Harbor New York, 1988), and proteins can be isolated from other cellular proteins by selective hybridization with antibodies. The protein can be purified to the desired degree by standard methods of protein purification (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold
(See Spring Harbor New York, 1989). The amino acid sequence of any protein, polypeptide or peptide of the invention can be inferred from the nucleic acid sequence or can be determined by sequencing of isolated or recombinantly produced proteins.

  The terms “peptide”, “polypeptide” and “protein” can be used interchangeably herein and refer to a polymer of amino acids and include full-length proteins and fragments thereof. As used herein and in the claims, “a” can mean one or more, depending on the context in which it is used. An amino acid residue is an amino acid formed by chemical digestion (hydrolysis) of a polypeptide at a peptide bond. The amino acid residues described herein are preferably in the L isomer form. However, residues in the D isomeric form can be replaced with any L amino acid residue as long as the desired functional properties are retained by the polypeptide. As used herein, standard polypeptide nomenclature (described in J. Biol. Chem., 243: 3552-59 (1969) and adopted in US Patent Enforcement Regulation 1,822 (b) chapter. ).

  As will be appreciated by those skilled in the art, the present invention also includes those polypeptides having minor changes in amino acid sequence or other properties. Amino acid substitutions can be selected by known parameters that are neutral (see, eg, Robinson WE Jr, and Mitchell WM, AIDS 4: S151-S162 (1990)). Such mutations can occur naturally as allelic variations (eg, due to genetic polymorphism) or human intervention (eg, mutagenesis of cloned DNA sequences (eg, induced points) Mutations, deletions, insertions and substitution mutations). Small changes in the amino acid sequence are usually preferred (eg, conservative amino acid substitutions, small internal deletions or insertions, and additions or deletions at the ends of the molecule). Substitutions can be designed, for example, based on the model of Dayhoff et al. (Atlas of Protein Sequence and Structure 1978, Nat'l Biomed. Res. Found., Washington, D.C.). These modifications can result in changes in the amino acid sequence, provide silent mutations, modify restriction sites, or provide other specific mutations. Similarly, such amino acid changes result in different nucleic acids encoding polypeptides and proteins. Accordingly, alternative nucleic acids are also contemplated by such modifications.

  The present invention also provides a cell containing the nucleic acid of the present invention. A cell containing a nucleic acid that encodes a protein is typically capable of replicating DNA and may also typically express the encoding protein. The cell can be a prokaryotic cell (especially for purposes of producing large amounts of nucleic acid) or a eukaryotic cell (especially a mammalian cell). For purposes of expressing the encoded protein such that the resulting produced protein has mammalian protein processing modifications, the cell is preferably a mammalian cell.

  The nucleic acids of the invention can be delivered to cells by any selected means, particularly depending on the purpose of delivery of the compound and target cells. Many delivery means are well known in the art. For example, liposomes in combination with electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes and a nuclear localization signal peptide for delivery to the nucleus can be utilized. This is well known.

  The present invention also contemplates that mutated cellular genes required for viral growth (produced by the present method) as well as cells containing these mutants may also be useful. These mutant genes and cells containing them can be isolated and / or produced using standard methods according to the methods described herein.

  It should be understood that the sequences shown herein may contain minor sequence errors. Such errors are corrected, for example, by using the hybridization procedure described above with various probes derived from the described sequences so that the coding sequence can be reisolated and resequenced. obtain.

  As described in the Examples, the present invention provides the knowledge of “serum survival factors” present in serum that are necessary for the survival of persistent virus-infected cells. Isolation and characterization of this factor indicates that this factor is a protein, has a molecular weight between about 50 kD and 100 kD, is resistant to inactivation in low pH (eg, pH 2) and chloroform extractions, about 5 It was shown to be inactivated by boiling for a minute and a low ionic strength solution (eg, about 10 mM to about 50 mM). Thus, the present invention provides a purified mammalian serum protein having a molecular weight between about 50 kD and 100 kD. This protein is resistant to inactivation at low pH and resistant to inactivation by chloroform extraction, inactivated when boiled, inactivated in low ionic strength solution, and persistently infected with reovirus Is selectively substantially inhibited from survival of cells persistently infected with reovirus. A factor consistent with the above physical characteristics is the addition of this factor to serum-free medium (previously unable to maintain the survival of persistent virus-infected cells) and this putative factor added. This can be easily demonstrated by determining whether the medium can now maintain persistent virus-infected cells, particularly cells that are persistently infected with reovirus. As used herein, “purified” protein means that the protein is at least pure enough that the approximate molecular weight can be determined.

  The amino acid sequence of the protein can be deduced by standard methods. For example, antibodies to proteins can be raised and used to screen expression libraries to obtain nucleic acid sequences that encode the proteins. This nucleic acid sequence is then simply translated into the corresponding amino acid sequence. Alternatively, a portion of the protein can be sequenced directly by standard amino acid sequencing methods (amino terminal sequencing). This amino acid sequence can then be used to generate a series of nucleic acid probes that contain all possible coding sequences for a portion of the amino acid sequence. A series of probes are used to screen the cDNA library, and the rest of the coding sequence, and thus ultimately the corresponding amino acid sequence, is obtained.

  The present invention also provides additional methods for detecting and isolating serum survival factors. For example, to determine whether any known serum component is required for virus growth, that known component can be inhibited or removed from the culture medium and persistently infected cells do not survive By determining whether or not virus growth is inhibited. The factor can be returned (or the inhibition removed) and it can be determined whether the factor allows the virus to grow.

  In addition, other, unknown serum components can also be found to be essential for virus growth. Serum is fractionated by a variety of standard techniques, and fractions are added to serum-free media to determine if factors exist in the reaction that allow growth previously inhibited by serum deletions. Can be done. The fraction with this activity can then be further fractionated until the factor is relatively free of other components. This factor can then be characterized by standard methods such as size fractionation, denaturation and / or inactivation by various means, and the like. Preferably, once this factor has been purified to the desired level of purity, it is added to cells in serum-free medium, conferring the ability to grow on viruses that did not grow in the case of serum-free medium alone. Is confirmed. This method can be repeated to confirm the requirement of a particular factor for any desired virus. This is because each serum factor found to be necessary for any one virus may be required for many other viruses. In general, the more closely related the virus and the more similar the mode of infection of the virus, the more likely the factors required for one virus are required for the other virus.

  The present invention also provides methods for treating viral infections utilizing our findings. The subject of any method described herein can be any animal, preferably a mammal such as a human, a livestock animal (eg, cat, dog, horse, pig, goat, depending on the virus). Sheep, or cattle), or research animals (eg, mice, rats, rabbits, or guinea pigs).

  The present invention provides a method of reducing or inhibiting and treating viral infection in a subject. This method involves subject to serum proteins described herein (ie, resistant to inactivation at low pH and resistant to inactivation by chloroform extraction, and inactivated when boiled. Between about 50 kD and 100 kD, preventing the survival of at least some of the cells persistently infected with the virus when inactivated in a low ionic strength solution and removed from a cell culture containing cells persistently infected with the virus Administering an inhibitory amount of a composition that inhibits the function of a serum protein having a molecular weight of, thereby treating a viral infection. The composition can include, for example, an antibody that specifically binds to a serum protein, or an antisense RNA that binds to RNA encoded by a gene that functionally encodes the serum protein.

  Any virus that can infect the selected subject to be treated can be treated by the method. As noted above, any serum protein or survival factor found by this method to be necessary for the growth of cells infected with any one virus is responsible for the growth of cells infected with many other viruses. It can be found necessary. For any given cell and virus combination, serum proteins or factors can be confirmed to be necessary for growth by the methods described in the present invention. Cellular genes identified by examples using reovirus, mammalian pathogens, and rat cell lines have general applicability to all known and other viral infections, including undiscovered human pathogens . This pathogen includes, but is not limited to: human immunodeficiency virus (eg, HIV-1, HIV-2); parvovirus; papilloma virus; hantavirus; influenza virus (eg, influenza A, B, Hepatitis viruses AG; astrovirus; rotavirus; coronavirus such as human respiratory coronavirus; picornavirus (eg, human rhinovirus and enterovirus); ebola virus; human herpesvirus (Eg, HSV-1-9); human adenovirus; hantavirus; for animals, the animal counterpart of any of the human viruses listed above, animal retroviruses (eg, simian immunodeficiency virus, avian immunodeficiency) Virus, bovine immunodeficiency Virus, feline immunodeficiency virus, equine infectious anemia virus, goats joint encephalitis virus Arena virus, arboviruses, tick-borne virus or visna virus).

The protein inhibitory amount of the composition is readily determined, for example, by administering various doses to the cell or subject and then adjusting the effective amount to inhibit the protein according to the amount or body weight of the subject's blood. Can be done. A composition that binds to a protein can be readily determined by running a putative binding protein on a protein gel and observing changes in the movement of the protein on the gel. Protein inhibition can be determined by any desired means, such as adding inhibitors to the complete medium used to maintain persistently infected cells and observing cell viability. The composition can include, for example, an antibody that specifically binds to a serum protein. Specific binding by an antibody means that the antibody can be used to selectively remove an agent from serum, or to inhibit the biological activity of the agent, and a radioimmunoassay (RIA), bioassay, or It means that it can be easily determined by enzyme linked immunosorbent (ELISA) technique. The composition can include, for example, an antisense RNA that specifically binds to an RNA encoded by a gene encoding a serum protein. Antisense RNA can be synthesized and used by standard methods (eg, Antisense RNA and DNA, edited by DA Melton, Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY (1988)).

  The method provides a method of screening for compounds that are effective in treating or preventing viral infection. The method comprises the step of administering a compound to a cell comprising a cellular gene that functionally encodes a gene product that is necessary for viral growth in the cell but not for cell survival, and the level of gene product produced and And / or including detecting activity (ie, function), the reduction or elimination of gene product and / or gene product activity is a method of indicating compounds for the treatment or prevention of viral infection. The cellular gene can be, for example, the nucleic acid shown below: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11 SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 23 SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, Sequence number 53, sequence number 54, sequence number 55, sequence number 56, sequence number 57, sequence number 58, sequence number 59, sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, Sequence number 78, sequence number 79, sequence number 80, sequence number 81, sequence number 82, sequence number 83, sequence number 84, sequence number 85, sequence number 86, sequence number 87, sequence number 88, sequence number 89, sequence number 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, Sequence number 114, Sequence number 115, Sequence number 116, Sequence number 117, Sequence number 118, Sequence number 119, Sequence number 120, Sequence number 121, Sequence number 122, Sequence number 123, Sequence number 124, Sequence number 125, Sequence number 126 or SEQ ID NO: 127 (sometimes referred to herein as Sequence Listing 2 for brevity), any homologs thereof, using the methods provided herein for obtaining such genes. Any other gene obtained. It will be appreciated that the cellular gene may be naturally present in the cell being screened or introduced into the cell with an appropriately expressed expression vector (well known in the art). The level of the gene product can be measured by any standard means (eg, by detection with an antibody specific for the protein). The level of the gene product can be compared to the level of the gene product in a control cell that has not been contacted with the compound. The level of gene product can be compared to the level of gene product in the same cell prior to addition of the compound. Activity or function can be measured by any standard method, such as an enzymatic assay that measures the conversion of a substrate to a product, or a binding assay that measures, for example, the binding of a protein to a nucleic acid. Examples of gene products disclosed herein whose activity / function can be measured include tristetraproline (human ZFP-36), 6-pyruvoyl-tetrahydropterin synthase, eukaryotic DnaJ-like protein, ID3 (Inhibitors of DNA binding 3), N-acetylglucose-aminyltransferase I (mGAT-1), cleavage stimulating factor (CSTF2), TAK1 binding protein, human zinc transcription factor ZPF207, Dlx2, Smad7 (Mad related protein) and Contains P-glycoprotein (mdr1b). The activity can be compared to the activity in control cells that have not been contacted with the compound, or the same cells prior to addition of the compound. Relatedly, the regulatory region of this gene can be operably linked to a reporter gene and compounds can be screened for inhibition of the reporter gene. Such reporter constructs can be described herein.

  The present invention also provides a method of screening for compounds that are effective in treating or preventing viral infection. This method comprises the steps of contacting this compound with a gene product of a cellular gene comprising the nucleic acid of Sequence Listing 2 or any homologue thereof, and detecting the function of the gene product, wherein the function reduction or deletion is virus It is a method that shows compounds that are effective in the treatment or prevention of infection. Examples of gene products disclosed herein that can be utilized in the methods of the invention include tristetraproline (human ZFP-36), 6-pyruvoyl-tetrahydropterin synthase, a eukaryotic DnaJ-like protein, ID3 (inhibitor of DNA binding 3), N-acetylglucose-aminyltransferase I (mGAT-1), cleavage stimulating factor (CSTF2), TAK1 binding protein, human zinc transcription factor ZPF207, Dlx2, Smad7 (Mad related protein) And P-glycoprotein (mdr1b).

  The present invention provides a method for selectively removing cells persistently infected with a virus from an animal cell culture that can survive for a first period in the absence of serum. The method comprises the step of growing a cell culture in the absence of serum during a second period in which persistently infected cells cannot survive in the absence of serum, whereby cells that are persistently infected with virus from the cell culture. Selectively removing. The second period should be shorter than the first period. Therefore, simply remove serum from a standard culture medium composition for a period of time (eg, by removing the serum-containing medium from the culture container, washing the cells, and returning the serum-free medium to the container), then The serum can be returned to the culture medium after the time of serum starvation. Alternatively, culture-derived serum survival factors can be inhibited instead of a serum starvation step. In addition, it may instead interfere with virus-factor interactions. Such virus removal methods can be performed periodically to ensure that the cultured cells remain free of virus. The duration of serum removal can vary widely, a typical range is from about 1 day to about 30 days; a preferred period can be from about 3 days to about 10 days, and a more preferred period is about 5 days. Day to about 7 days. This period has the ability of certain cells to survive in the absence of serum as well as the life cycle of the target virus (eg, about 24 hours for reovirus, and the three-day starvation of cells is dramatic. Provide a result).

  Furthermore, the time period can also be shortened by passaging the cells during starvation; in general, the increase in the number of passages requires the serum starvation (in order to obtain complete removal of virus from the culture ( Or the time of serum factor inhibition) may be reduced. During passage, the cells are typically exposed to serum for a short time (typically about 3 to about 24 hours). This exposure both stops the action of the trypsin used to remove the cells and stimulates the cells to another growth cycle, thus assisting this selection process. Thus, the starvation / serum cycle can be repeated to optimize the selection effect. Other standard culture parameters (eg, culture confluence, pH, temperature, etc.) may be varied to alter the time required for serum starvation (or serum survival factor inhibition). This period is simply removed for various periods of time for any given viral infection, and then the culture is tested for the presence of infected cells at each test period (eg, by virtue of the ability to survive in the absence of serum). , And confirmed by standard virus titration and immunohistological techniques by quantifying the virus in the cells), and then detecting in which period of serum deficiency the virus-infected cells were removed, It can be easily determined. It is preferred that a shorter serum deficiency period is used that still provides for the removal of persistently infected cells. In addition, the cycle of starvation, then re-addition of serum, and determination of the amount of virus remaining in the culture can be repeated until no infected cells are actually present in the culture.

Thus, the method can further include passaging the cells (ie, transferring the cell culture from the first container to the second container). Such migration can facilitate selective loss of survival of virus-infected cells. The movement can be repeated several times. Migration is achieved by standard methods of tissue culture (eg, Freshney, Culture of Animal Cells, A Manual of Basic Technique, 2nd edition, Alan R. Liss, Inc.,).
New York, 1987).

  The method further provides a method for selectively removing cells persistently infected with the virus from the cell culture. This method is resistant to inactivation at low pH and resistant to inactivation by chloroform extraction, inactivated when boiled, inactivated in low ionic strength solution, and persistently infected with reovirus In the absence of a functional form of a serum protein having a molecular weight between about 50 kD and 100 kD that substantially prevents the survival of cells persistently infected with reovirus when removed from a cell culture containing cells. Growing the cell culture. The absence of a functional form may be due to any of several standard means (eg, binding the protein to an antibody selective for that protein (either before or after the serum is added to the cells). Binding to the antibody in the serum; if prior to addition, the serum protein can be removed from the serum by, for example, binding the antibody to the column and passing the serum through the column, and then the live protein Or a compound that inactivates the protein, or a compound that interferes with the interaction between the virus and the protein).

  Accordingly, the present invention provides a method for selectively removing cells that are persistently infected with viruses from cell cultures grown in serum-containing media. This method is resistant to inactivation at low pH and resistant to inactivation by chloroform extraction, inactivated when boiled, inactivated in low ionic strength solution, and persistently infected with reovirus Inhibiting a protein having a molecular weight between about 50 kD and 100 kD in serum that substantially inhibits the survival of cells persistently infected with reovirus when removed from a cell culture containing the cells. . Alternatively, the interaction between the virus and serum protein can be disrupted and cells persistently infected with the virus can be selectively removed.

  Any virus capable of some form of persistent infection can be removed from the cell culture using the removal methods of the present invention. This method involves the removal, inhibition or otherwise obstruction of serum proteins (eg, as exemplified herein) and is also necessary for viral growth but not essential for cells. Including removal, inhibition or otherwise interference of gene products from any cellular gene found by the method of the invention. For example, a DNA virus or RNA virus can be targeted. Whether cells infected with the selected virus can be selectively removed from the culture by removal of serum was selected by starving (or inhibiting serum survival factors) the virus-permissive cell serum. By adding virus to cells, adding serum to the culture, and observing whether infected cells die (ie titer the level of virus in living cells with antibodies specific for the virus) Can be easily determined.

  Any animal cell culture that can be maintained for a period of time in the absence of serum (ie, any cell that is typically grown and maintained in culture in serum) can be utilized using the methods of the present invention. It can be purified from a viral infection. For example, primary cultures and established cultures and cell lines can be used. In addition, cultures of cells from any animal and from any tissue or cell type in that animal that can be cultured and maintained for a period of time in the absence of serum can be used. For example, typically a culture of cells from a tissue infected with an infectious virus and particularly persistently infected can be used.

  As used in the claims of the present application, “in the absence of serum” means a level at which persistently virus-infected cells do not survive. Typically, the threshold level is about 1% serum in the medium. Thus, about 1% or less of serum can be used, for example, about 1%, 0.75%, 0.50%, 0.25%, 0.1% serum, or serum-free.

  As used herein, “selectively destroying” cells that are persistently infected with a virus means that substantially all of the cells that are persistently infected with the virus die, resulting in removal. It means that the presence of virus-infected cells cannot be detected in the culture immediately after the procedure is performed. Furthermore, “selectively discard” includes that cells not infected with the virus are generally not killed by this method. Some viable cells can still produce virus, albeit at a low level, and some may have incomplete pathways that lead to death by the virus. Typically, more than about 90% of the culture, and more preferably about 95%, 98%, 99%, or 99.99%, to kill substantially all cells persistently infected with the virus. More than 99% of virus-containing cells die.

The method also provides a nucleic acid comprising the regulatory region of any gene. Such regulatory regions can be isolated from genomic sequences isolated and sequenced as described above, and by any observed characteristics characteristic of that type of regulatory region, and the coding region of the gene Can be identified by their start codons and their relationship. The invention also provides a construct comprising a regulatory region operably linked to a reporter gene. Such constructs can be made by routine subcloning methods, and many vectors are available, in which regulatory regions can be subcloned upstream of the marker gene. Marker gene detection of marker gene product Accordingly, the method also provides a method of screening a compound for treating viral infection, comprising any of the nucleotide sequences shown in Sequence Listing 2, or their Administering the compound to a cell containing any of the above constructs comprising one regulatory region of a gene comprising any homolog (its inhibition or reduction in expression causes inhibition of viral replication), wherein The region is operably linked to the reporter gene) and detecting the level of reporter gene product produced, the reduction or elimination of the reporter gene product being used to treat the viral infection Compounds are shown. The compound detected by this method inhibits the transcription of the gene from which the regulatory region is isolated, and thus inhibits the production of the gene product produced by the gene in treating the subject, and thus the virus. Treat the infection.

  Some genes, when disrupted by the inventive method of retroviral insertion, resulted in overexpression of the gene product and this overexpression inhibited viral replication. Accordingly, the present invention provides a method of screening a compound for efficacy in the treatment of viral infections, wherein the method comprises treating the compound with an intracellular gene (its overexpression inhibits viral replication, but the cellular And a step of detecting the level of the gene product produced, wherein the increase in the gene product treats the virus infection Compounds that are effective to do Typically, the increase is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, relative to the gene product produced in the absence of the compound. An increase of 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or more.

  The present invention further provides a method of reducing or inhibiting viral infection in a subject, the method comprising expressing or functioning a gene product encoded by a gene comprising a nucleic acid set forth in any of Sequence Listing 2 or a homologue thereof. Administering to the subject an amount of the composition that inhibits crystallization, thereby treating the viral infection. Reduction or inhibition of viral infection is, of course, the initial infection of the subject and the infection of uninfected cells (eg, inhibiting viral replication in the subject's cells) in an already infected subject. And both. The composition can include, for example, an antibody that binds to the protein encoded by the gene. The composition can also include an antibody that binds to the receptor for the protein encoded by the gene. Such antibodies can be raised against proteins selected by the standard methods described above and can be either polyclonal or monoclonal, with monoclonal being preferred. Alternatively, the composition can include an antisense RNA that binds to the RNA encoded by the gene, as described above. Examples of antisense RNA that are therapeutically useful include fragments of the nucleic acids described above. Further, the composition can include a nucleic acid that functionally encodes an antisense RNA that binds an RNA encoded by the gene. Other useful compositions will be readily apparent to those skilled in the art.

  The present invention also provides a method of treating a viral infection in a subject, wherein the method provides to the subject a therapeutically effective amount that increases the expression of a gene (its overexpression reduces or inhibits viral replication). Administering a composition of: Typically, the increase is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to the gene product produced in the absence of the composition. , 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%, or more.

  The present invention further provides a method of reducing or inhibiting viral infection in a subject, wherein the method comprises, for example, subjecting a nucleic acid shown in Sequence Listing 2 in a cell selected from, for example, a subject or a homogeneous source. An endogenous gene containing (inhibiting or reducing its expression results in inhibition of viral replication) or a homolog thereof, a genetic form incapable of producing a functional gene product of that gene, or a reduction in the functional gene product of that gene Mutating ex vivo to a genetic form that produces the amount obtained, and returning the cell back into the subject (or replacing in the case of the subject's own cell), thereby Reduce viral infection of cells in a subject. Cells can be selected according to representative target cells of a particular virus whose infection is reduced, prevented, or inhibited. Preferred cells for some viruses are hematopoietic cells. When the selected cell is a hematopoietic cell, a virus whose infection can be reduced or inhibited can include, for example, HIV (including HIV-1 and HIV-2). However, many other virus-cell combinations will be apparent to those skilled in the art.

  The invention also encompasses a method of reducing or inhibiting viral infection in a subject, which method comprises, for example, selecting a nucleic acid set forth in Sequence Listing 2 in a selected cell from a subject or from a homogeneous source. Exogenously mutating an endogenous gene (its overexpression causes inhibition of viral replication) or a homolog thereof to a genetic form that expresses the gene at a higher level than the endogenous gene, and Placing the cells in the subject or replacing the cells of the subject. Typically, higher levels are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% over endogenous genes that are not mutated. 125%, 150%, 175%, 200%, 300%, 400%, 500%, or more. The cells can be selected according to a representative target cell of the particular virus whose infection is to be reduced, prevented or inhibited. Preferred cells for some viruses are hematopoietic cells. When the selected cells are hematopoietic cells, viruses that can be reduced or inhibited from infection can include, for example, HIV (including HIV-1 and HIV-2). However, many other virus-cell combinations will be apparent to those skilled in the art.

  The present invention further provides a method of increasing resistance to viral infection in a subject, which method comprises, for example, selecting a nucleic acid shown in Sequence Listing 2 in a selected cell from a subject or from a homogeneous source. A process wherein an endogenous gene (inhibiting or reducing its expression increases resistance to viral infection) is mutated ex vivo, the endogenous gene being unable to produce a functional gene product of the gene Mutated to a genetic form produced to produce a reduced gene form, or a reduced quantity of a functional gene product of the gene, and to place the cell in the subject, whereby the test Increases resistance of cells to viral infection in the body. The virus can be HIV, particularly if the cell is a hematopoietic cell. However, many other virus-cell combinations will be apparent to those skilled in the art.

  Furthermore, the present invention provides a method for the isolation of cellular genes utilized in tumor progression. The present invention provides a method for identifying a cellular gene that can suppress a malignant phenotype in a cell, comprising: (B) selecting a cell that expresses the marker gene, and (c) a cellular gene into which the marker gene has been inserted in soft agar or Matrigel. Identifying a gene that can be isolated from selected cells that can grow in, thereby suppressing a malignant phenotype in the cell. This method can be performed using any selected non-transformed cell line, many of which are known in the art.

  The present invention further provides a method for identifying a cellular gene capable of suppressing a malignant phenotype in a cell, wherein the method comprises: (a) a vector encoding a selectable marker gene lacking a functional promoter; (B) selecting a cell that expresses the marker gene, and (c) isolating the cellular gene into which the marker gene has been inserted from the selected and transformed cells. And thereby identify genes that can suppress the malignant phenotype in the cell. An untransformed phenotype can be determined by any number of standard methods in the art, exemplified by the inability to grow in soft agar, or in Matrigel.

  The present invention further provides a method of screening for a compound for inhibiting a malignant phenotype in a cell, which comprises a cellular gene that functionally encodes a gene product involved in establishing a malignant phenotype in the cell. The method includes administering the compound to a containing cell and detecting the level of the gene product produced, wherein reduction, inhibition or elimination of the gene product indicates a compound that is effective for suppression of the malignant phenotype. Detection of the level or amount of gene product produced can be any of several methods standard in the art (e.g., assayed for protein level or amount and selected based on the particular gene product (e.g. , Protein gels, antibody-based assays, detection of labeled RNA) or directly.

  The present invention also provides a method of screening for compounds to suppress a malignant phenotype in a cell, which function gene product (its overexpression is involved in the suppression of the malignant phenotype in the cell). Administering the compound to a cell containing a cellular gene that encodes the gene, and detecting the level of the gene product produced, wherein the increase in the gene product suppresses a malignant phenotype Are effective compounds.

  The present invention further provides a method of suppressing a malignant phenotype in a cell of a subject, comprising: subjecting the subject to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, It is found that the nucleic acid gene shown in SEQ ID NO: 29, SEQ ID NO: 36, or SEQ ID NO: 94, or a gene containing these homologs, or overexpression thereof is involved in suppressing the malignant phenotype of the cell. Administering an amount of the composition that inhibits the expression or functionalization of the gene product encoded by any gene (eg, a clone designated herein with an “x”), whereby malignant expression Suppress the type. The composition may alternatively include an antibody that binds to the protein encoded by the gene. As another example, the composition can include an antibody that binds to a receptor for the protein encoded by the gene. The composition can include an antisense RNA that binds to an RNA encoded by the gene. Further, the composition can include a nucleic acid that functionally encodes an antisense RNA that binds to an RNA encoded by the gene.

  The present invention further provides a method of suppressing a malignant phenotype in a cell of a subject, wherein the method comprises expressing to the subject the expression of a gene product whose overexpression is involved in suppressing the malignant phenotype in the cell Administering an increasing amount of the composition. The gene product is the product of a gene whose destruction of the upstream gene by the vector of the present invention results in the overexpression of the downstream gene, and the overexpression of the downstream gene demonstrates the transformed phenotype. possible. The composition can be, for example, an inhibitor of a COX2 enzyme (eg, a small molecule inhibitor).

  The diagnostic or therapeutic agent of the present invention can be any standard means for administering a therapeutic or diagnostic agent to its selected site, or a standard method for administering that type of functional entity. Can be administered to a subject or animal model. For example, the drug can be administered orally, parenterally (eg, intravenously), by intramuscular injection, by intraperitoneal injection, topically, transdermally, and the like. The drug can be administered, for example, as a complex with cationic liposomes or encapsulated in anionic liposomes. The composition can include various amounts of the selected agent in combination with a pharmaceutically acceptable carrier, and can further include other pharmaceutical agents, agents, carriers, adjuvants, diluents, and the like, if desired. Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or in any conventional form as an emulsion. Depending on the mode of administration, the drug can be optimized, eg, by encapsulation, to avoid degradation in the subject.

  Dosing is typical for the administration of antiviral or anti-cancer agents and is used in administration, depending on the mode of administration, the disease or condition being treated, and the condition of the individual subject. . Dosing also depends on the composition being administered (eg, protein or nucleic acid). Such dosing is known in the art. Moreover, dosing can be adjusted according to representative dosing for the particular disease or condition being treated. Further, the viral titer in the target cell type cultured cells can be used to optimize dosing for the target cells in vivo and changes achieved in the same type of cultured cells as the target cell type Transformation from dosing can be monitored. Often a single dose may be sufficient; however, the dose may be repeated if desired. The medication should not be so large as to cause harmful side effects. In general, dosing will vary depending on the age, condition, sex, and degree of disease for the subject and can be determined by one skilled in the art. Medication can also be adjusted by the individual physician in the case of any complication.

  For administration to cells in a subject, the composition will naturally adapt to the body temperature of the subject once it is in the subject. For ex vivo administration, the composition is added by any standard method that maintains cell viability (eg, the composition is added to the cell culture medium (appropriate for the target cells) and the medium is applied directly to the cells. Can be administered). As is known in the art, any medium used in this method can be aqueous and non-toxic so as not to render the cells non-viable. In addition, the medium can contain standard nutrients to maintain cell viability, if desired. For in vivo administration, the complex can be added, for example, to a blood or tissue sample from a patient, or to a pharmaceutically acceptable carrier (eg, saline and buffered saline) and the art Can be administered by any of several means known in Examples of administration include parenteral administration (eg, intravenous injection, including local perfusion through blood vessels that deliver blood to tissues or organs with target cells, or aerosol inhalation, subcutaneous injection or intramuscular injection), topical administration ( (Eg, to skin wounds or injuries), eg, direct transfection into bone marrow cells prepared for transplantation and subsequent transplantation into a subject, and organ (subsequently transplanted into the subject) Direct transfection may be mentioned. Additional methods of administration include oral administration (especially when the composition is encapsulated) or rectal administration (especially when the composition is in suppository form). Pharmaceutically acceptable carriers include any substance that is not biologically or otherwise undesirable, i.e., the substance causes any undesirable biological effect or is pharmaceutically acceptable. It can be administered to an individual with the selected complex without interacting in a deleterious manner with any other component of the composition in which it is contained.

  In particular, if a particular cell type is targeted in vivo, for example by local perfusion of an organ or tumor, cells from the target tissue can be biopsied and optimal for transfer of the complex to that tissue Dosing can be determined in vitro as described herein and known in the art to optimize in vivo dosing (including concentration and length of time). Alternatively, cultured cells of the same cell type can also be used to optimize dosing for target cells in vivo.

  For either ex vivo or in vivo use, the complex can be administered at any effective concentration. An effective concentration is an amount that results in a reduction, inhibition, or prevention of viral infection or a reduction or inhibition of the transformed phenotype of the cell.

  The nucleic acid can be administered by any number of means, which can be selected according to the vector utilized, the organ or tissue to be targeted (if any), and the characteristics of the subject. The nucleic acid can be administered systemically, such as intravenous, intraarterial, oral, parenteral, subcutaneous, if desired in a pharmaceutically acceptable carrier such as saline. Nucleic acids can also be administered by direct injection into an organ or by injection into a blood vessel that delivers blood to a target tissue. For infection of lung or tracheal cells, it can be administered intratracheally. The nucleic acid can further be administered topically, transdermally, and the like.

  The nucleic acid or protein can be administered in the composition. For example, the composition can include other pharmaceutical agents, drugs, carriers, adjuvants, diluents, and the like. Furthermore, the composition can include, in addition to the vector, lipids such as liposomes (eg, cationic liposomes (eg, DOTMA, DOPE, DC-cholesterol) or anionic liposomes). Liposomes can further include proteins to facilitate the targeting of specific cells, if desired. Administration of a composition comprising a vector and cationic liposomes can be administered to blood transfused into a target organ or can be inhaled into the airway to target airway cells. Regarding liposomes, see, eg, Brigham et al., Am. J. et al. Resp. Cell. Mol. Biol. 1: 95-100 (1989); Felgner et al., Proc. Natl. Acad. Sci USA 84: 7413-7417 (1987); U.S. Pat. No. 4,897,355.

  For viral vectors that include nucleic acids, the composition can include a pharmaceutically acceptable carrier such as phosphate buffered saline or saline. Viral vectors can be selected according to the target cell, as is known in the art. For example, adenoviral vectors, particularly replication defective adenoviral vectors, can be utilized to target any number of cells because of their broad host range. Many other viral vectors are available and their target cells are known.

  Throughout this application, various publications are cited. The disclosures of these publications are incorporated herein by reference in their entirety to more fully describe the state of the art to which this invention belongs.

(Selective exclusion of virus-infected cells from cell culture)
Rat intestinal cell line-1 cells (RIE-1 cells) were typically grown in Dulbecco's modified Eagle medium (high glucose) supplemented with 10% fetal calf serum. To start the experiment, cells continuously infected with reovirus are grown to near confluence, then the medium is removed, the cells are washed in PBS, and returned to flask medium without added serum. Serum was removed from the growth medium by Typically, the serum content was reduced to 1% or less. Cells are serum starved for a period of several days or about a month to bring them into quiescence or growth arrest. A medium containing 10% serum is then added to the quiescent cells to stimulate cell growth. Surviving cells are sustained by immunohistochemical techniques (sensitivity of 1 infectious virus per ml of homogenized cells) used to verify whether the cells contain any infectious virus. It is found that the cells are not infected.

(Isolation of cell genomic DNA)
Gene Trap Library: A library is generated by infecting RIE-1 cells with a retroviral vector (U3 gene-trap) at a ratio of less than 1 retrovirus per 10 cells. When the U3 gene trap retrovirus is integrated into a gene that is actively transcribed, the neomycin resistance gene encoded by the U3 gene trap retrovirus is also transcribed, which confers resistance to the antibiotic neomycin. Cells with a gene trap event can survive exposure to neomycin, but cells without a gene trap event die. The various cells that survive the neomycin selection are then expanded as a library of gene trap events. Such a library can be generated using any retroviral vector that has the property of expressing a reporter gene from a transcriptionally active cellular promoter that tags the gene for later identification.

Reovirus selection: Reovirus infection is typically lethal to RIE-1 cells, but can result in the development of persistently infected cells. These cells continue to grow while producing infectious reovirus particles. For the identification of gene trap events that confer reovirus resistance to cells, persistently infected cells must be eliminated or they are recorded as false positives. The inventors have found that RIE-1 cells persistently infected with reovirus are very poorly resistant to serum starvation, passage, and plating at low density. Accordingly, the inventors have developed a protocol for screening an RIE-1 gene trap library that selects both reovirus sensitive cells and cells persistently infected with reovirus.
1. RIE-1 library cells are grown to near confluence and the serum is then removed from the medium. Cells are starved for serum for several days to bring them to rest or growth arrest.
2. Library cells are infected with reovirus at a reovirus titer greater than 10 per cell and serum starvation is continued for several more days.
3. Infected cells are passaged (the process in which they are exposed to serum for 3-6 hours) and then starved for serum for an additional few days.
4). The surviving cells are then grown in the presence of serum until a colony that is visible at the time they are cloned by limiting dilution develops.
Medium: Dulbecco's Modified Eagle Medium (High Glucose) (DME / HIGH) Hyclone Laboratories Cat. No. SH30003.02. Neomycin: An antibiotic used to select cells that do not have a U3 gene trap retrovirus (eg, from GENETICIN Sigma, catalog number G9516).
Rat intestinal cell line-1 cells (RIE-1 cells): These cells are from the laboratory of Dr. Ray Dubois (VAMC). They are typically cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum.
Reovirus: Use either serotype 1 or serotype 3 laboratory strains. These were originally Bernard N. Obtained from Fields laboratory. These viruses have been described in detail.
Retrovirus: The U3 gene trap retrovirus used herein was developed by Dr. Earl Ruley (VAMC) and this library was generated using the general protocol suggested by him.
Serum: Fetal calf serum Hyclone Laboratories catalog number A-1115-L.

(Identification of tags for isolated nucleic acids)
Genomic sequences tagged with a vector (eg, U3 gene trap vector) are given numbers corresponding to the genomic library of mutant cells from which the sequence was isolated, and letters indicating unique members of the library. It is done. More than one sequence with the same number and letter indicates multiple unique sequences taken from the genome surrounding the vector insert that “tagged” the gene. Such genomic sequences are obtained using vector-based primers from which sequencing will occur from 3 ′ to 5 ′ or from 5 ′ to 3 ′. In the former case, the sequence derived from the vector primer must be reversed and complemented to restore the orientation of the gene inserted into the vector. Such a reverse complement sequence is designated “rE”. In the case of genomic sequencing from primers that occur 5 ′ to 3 ′ (ie, when the primer is the 3 ′ end of the vector) no change is required. This is because the derived sequence is that found in the disrupted gene. Such a sequence is designated “B4”. The homology shown below indicated that each genomic sequence was positive unless it was clearly shown that it was on the negative strand. By way of example, SEQ ID NO: 27 comprises a nucleic acid sequence that encodes a novel polypeptide on the positive strand, while the negative strand encodes ferritin.

（ウイルス感染のために必要な遺伝子）
本明細書中で開示される単離された配列のいくつかは、以下のタンパク質をコードする配列を含む：トリステトラプロリン（ｔｒｉｓｔｅｔｒａｐｒｏｌｉｎ）（ヒトＺＦＰ−３６）、６−ピルボイルテトラヒドロプテリンシンターゼ、真核生物のＤｎａＪ−様タンパク質、ＩＤ３（ＤＮＡ ｂｉｎｄｉｎｇ ３のインヒビター）、Ｎ−アセチルグルコサミニルトランスフェラーゼＩ（ｍＧＡＴ−１）、切断刺激因子（ＣＳＴＦ２）、ＴＡＫ１結合タンパク質、ヒトジンク転写因子ＺＰＦ２０７、Ｄｌｘ２、Ｓｍａｄ７（Ｍａｄ関連タンパク質）、およびＰ−糖タンパク質（ｍｄｒ１ｂ）。

(Gene required for virus infection)
Some of the isolated sequences disclosed herein include the following protein-encoding sequences: tristetraproline (human ZFP-36), 6-pyruvoyl tetrahydropterin synthase, true Nuclear DnaJ-like protein, ID3 (inhibitor of DNA binding 3), N-acetylglucosaminyltransferase I (mGAT-1), cleavage stimulating factor (CSTF2), TAK1-binding protein, human zinc transcription factor ZPF207, Dlx2, Smad7 (Mad related protein), and P-glycoprotein (mdr1b).

(Isolation of cellular genes that suppress malignant phenotype)
We utilized a gene capture method to select a cell line with a transformed phenotype (potentially a tumor cell) from a population of untransformed cells (RIE-1 parent). Its parent cell line, RIE-1 cells, does not have the ability to grow in soft agar or to produce tumors in mice. Following gene capture, cells were screened for their ability to grow in soft agar. These cells were cloned and the genomic sequence was obtained 5 ′ or 3 ′ of the retroviral vector (ie, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 36, or SEQ ID NO: 94; the sequence designated by “x” in the clone name). All of the cell lines behave as if they are tumor cell lines. Because they also induce tumors in mice.

  Two of the cell lines are associated with amplified expression of prostaglandin synthetase gene II or COX2. It has been shown that disruption of the gene function of the upstream gene by retroviral targeting led to increased expression of the downstream gene product COX2. Addition of a small molecule inhibitor of the COX2 enzyme resulted in reversion of the transformed phenotype. The COX2 gene is elevated in precancerous adenomas in humans and has been found to be overexpressed in human colon cancer. Inhibitors of COX2 expression also block tumor growth. One of the cell lines, x18 (SEQ ID NO: 94), has a disrupted gene that is currently represented in the EST (dbest) database, but this gene is not known (not present in GenBank).

  Each gene from which the provided nucleotide sequence is isolated (and all of the clones designated by “x”) represents a tumor suppressor gene. The mechanism by which the disrupted gene can suppress the transformed phenotype is currently unknown. However, each gene represents a tumor suppressor gene that is potentially unique because there is no genomic sequence corresponding to a known gene. The ability to rapidly select a tumor suppressor gene may provide a unique target in the process of treating or preventing cancer (potential for diagnostic testing).

(Isolation of whole genome gene)
An isolated nucleic acid of the invention (the sequence of which is set forth in any of SEQ ID NO: 1 to SEQ ID NO: 127), or smaller fragments thereof, are labeled with a detectable label and used as a probe. The rat genomic library (λ phage or yeast artificial chromosome vector library) is screened under high stringency conditions (ie, high salt concentration and high temperature to produce hybridization and wash temperature 5-20 ° C.). Clones were isolated and sequenced by standard Sanger dideoxynucleotide sequencing methods. Once the entire sequence of the new clone was determined, it was aligned with the probe sequence and its orientation relative to the probe sequence was determined. Second and third probes were each designed using sequences from either end of the combined genomic sequence. These probes were used to screen the library, isolate a new clone, and sequence this clone. These sequences are aligned with previously obtained sequences and new probes designed to correspond to either end sequence, and the entire process is repeated until the entire gene is isolated and mapped It was. If one end of the sequence cannot isolate a new clone at all, a new library can be screened. The complete sequence contains a regulatory region at the 5 ′ end and a polyadenylation signal at the 3 ′ end.
(Isolation of cDNA)
An isolated nucleic acid comprising an open reading frame (the sequence of which is set forth in any of SEQ ID NO: 1 to SEQ ID NO: 127), or smaller fragments thereof, or additional fragments obtained from a genomic library Label with a detectable label and use as a probe to screen some of the fragments of the invention and screen the cDNA library. A rat cDNA library obtains rat cDNA; a human cDNA library obtains human cDNA. As described above, repeated screening can be used to obtain complete coding sequences of genes from several clones if necessary. The isolate can then be sequenced and the nucleotide sequence determined by standard means (eg dideoxynucleotide sequencing).

(Isolation and characterization of serum survival factors)
The lack of resistance to serum starvation is due to the acquired dependence of persistently infected cells for serum factors (survival factors) present in the serum. Serum survival factor for persistently infected cells has a molecular weight between 50-100 kD and resists inactivation in low pH (pH 2) and chloroform extractions. It is inactivated by boiling for 5 minutes [once fractionated from whole serum (50-100 kD fraction)] and placed in a low ionic strength solution [10-50 mM].

Factors were sized using a centriprep molecular cut-off filter (Millipore and Amicon) having an exclusion size of 30-100 kd, and a dialysis tube (dialysis) having a molecular exclusion of 50 kd
The serum was isolated from the serum. Polyacrylamide gel electrophoresis and silver staining were used to determine that all obtained materials were between 50 and 100 kd. This confirms the validity of the initial isolation. Further purification was performed using ion exchange chromatography, heparin sulfate adsorption column, followed by HPLC. The pH of the serum fraction (30-100 kd fraction) was prepared using HCl at different pH conditions and the activity was determined after readjusting the pH to pH 7.4 prior to assessment of biological activity. The fraction containing the activity is dialyzed against a low ionic strength solution for various lengths of time and the ionic strength is restored to physiological conditions before determining biological activity by dialyzing the fraction against the medium. By adjusting, the sensitivity of low ionic strength was determined. Biological activity is maintained in the aqueous solution after chloroform extraction, indicating that the factor is not a lipid. Biological activity was lost after placing the 30-100 kd fraction in a 100 ° C. water bath for 5 minutes.

(Isolated nucleic acid molecule)
Isolated tagged genomic DNA was sequenced by standard methods using the Sanger dideoxynucleotide sequencing method. The sequence was performed on a computer data bank in a homology search. These genes can be therapeutic targets, especially because disruption of one or both alleles results in viable cells.

(Sequence Listing)

Claims (1)

An isolated nucleic acid comprising the nucleotide sequence set forth below:
Sequence number 1, Sequence number 2, Sequence number 3, Sequence number 5, Sequence number 6, Sequence number 7, Sequence number 8, Sequence number 9, Sequence number 10, Sequence number 11, Sequence number 12, Sequence number 13, Sequence number 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 6 , SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 80 SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97 , SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 112, Sequence 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 27.